The HVS in collaboration with GlaxoSmithKline (GSK), Rixensart, Belgium, developed several candidate live attenuated HAV vaccines. One such candidate was modified to become the currently licensed GSK inactivated HAV vaccine. In addition, the HVS developed a candidate recombinant hepatitis E vaccine that was highly promising and that completed phase II clinical trials. In studies to characterize this candidate hepatitis E vaccine, we performed extensive pre-clinical trials to determine the potency of the vaccine, the duration of protection, the optimum regimen for administration, its protective efficacy against homologous versus heterologous virus strains, its ability to prevent infection as well as hepatitis and the minimum antibody titer that was effective in preventing infection and hepatitis, respectively. In the clinical trial, the vaccine was 96% efficacious in preventing hepatitis E following three doses of vaccine and 87% efficacious following two doses. The vaccine had no detectable side effects. These results are outstanding for a vaccine. However, GSK elected not to pursue the manufacture of this vaccine and the NIAID is exploring other options for licensing the intellectual property for a hepatitis E vaccine. Several Asian pharmaceutical companies have expressed an interest in making a hepatitis E vaccine, perhaps in conjunction with a hepatitis A vaccine. The study of HCV, including vaccine development, is complicated by the genetic heterogeneity of the virus, which has resulted in the recognition of at least six major genotypes and many subtypes. Detecting and quantifying the virus requires different sets of primers for PCR amplification. We developed real-time PCR assays for the six major genotypes and have compared their specificity and sensitivity with other published assays. In addition, in FY 2009 and FY 2010, we further characterized the six major genotypes of HCV by preparing chimpanzee-derived plasma pools for each and determining the infectivity titer of them by reverse titration in additional chimpanzees. We are distributing these pools to the scientific community for vaccine studies and anticipate that they will be useful in furthering hepatitis C vaccine development. As an extension of these studies and as an aid to furthering basic research on HCV, we are preparing infectious cDNA clones of those HCV genotypes for which such clones are not available (genotypes 3, 4, 5 and 6). These studies are being performed in collaboration with Dr. Jens Bukh and Dr. William Satterfield. Considerable progress was made in 2010: infectious clones of genotypes 3 and 4 were completed and their infectivity confirmed by percutaneous intrahepatic transfection of chimpanzees. Less progress has been made with HCV genotypes 5 and 6. Despite repeated transfection attempts, neither of these full-length clones was infectious in chimpanzees. We are currently re-examining sequence data and attempting to replace parts of the clones that are suspected of being defective. Attempts to demonstrate infectivity of modified clones is being carried out at Bioqual Inc. Immunity to HCV is poorly understood and there is controversy over how complete and how long-lasting immunity to prior exposure of the virus is. This has implications for preventing chronic infections and their sequelae, cirrhosis and liver cancer. We have demonstrated that immunity to repeated exposure to HCV is genotype-specific, incomplete and relatively short-lived: even reexposure to the homologous virus can result in persistent infection. In other studies, GB virus-B (GBV-B) immunity was further studied. GBV-B is the closest relative to hepatitis C virus, which is very difficult to study because it is transmissible only to chimpanzees. In contrast, GBV-B is transmissible to tamarins, a species of New World monkey not considered to be endangered. Tamarins that had previously been infected with GBV-B were re-challenged with the same virus and shown to be immune to reinfection; this immunity was long-lived and apparently more complete than immunity to HCV in chimpanzees and humans. In additional studies to further characterize the similarity between infections by HCV and by GBV-B, in FY 2009 and FY 2010, we examined the mutation rate of acute, self-limiting versus chronic GBV-B infections in tamarins and demonstrated genetic changes similar to those observed in HCV infections. To date, liver cancer has not been observed in chronic GBV-B infections, perhaps because chronic infections are so uncommon, but we will monitor any additional chronically infected animals for evidence of liver cancer. In 2008, in collaboration with Michael Houghton (formerly of Chiron Corp.), we tested sera from the Chiron HCV vaccine trials in chimpanzees. Specifically, we tested serial sera from chimpanzees that had received Chiron's E1-E2 dimer vaccine (derived from the HCV-1 strain) that had been expressed from mammalian cells. Testing was performed with pseudo-typed retrovirus particles bearing the envelope glycoproteins of each of the major HCV genotypes (HCVpp). The magnitude of the antibody response against HCVpp bearing the envelope glycoproteins of a heterologous genotype 1a strain (H77C) paralleled the degree of protection of the chimpanzees: those animals with a high titer of neutralizing antibody against HCVpp were protected, whereas those with a lower titer were not. In addition, the neutralizing antibody was broadly cross-reactive, also neutralizing principally HCVpp bearing envelope glycoproteins of HCV genotypes 4a, 5a and 6a as well as those bearing the envelope glycoproteins of genoytpe 1a. We also tested in chimpanzees an HCV vaccine consisting of expressed HCV E1 protein, either with or without expressed HCV NS3 protein. The vaccine was manufactured by Innogenetics N.V. and was tested under a Cooperative Research and Development Agreement (CRADA) with the company. In contrast to the experience with the Chiron vaccine, none of the chimpanzees was protected from infection or from persistent infection and none had neutralizing antibody to HCV as measured with the HCVpp test. The decisive results of this study led to the abandonment of the E1 vaccine by Innogenetics. Also in 2010, we developed the chimpanzee model for norovirus infection and vaccine development. Susceptibility of chimpanzees to Norwalk virus was reported in 1978, when oral infection was documented. However, the animal model was not utilized again until recently. Using the same inoculum as used in the earlier studies, we could infect chimpanzees orally but only with very high doses of virus. We demonstrated that it was more practical to infect them intravenously, a procedure that has been used extensively for infections of nonhuman primates with hepatitis A virus and hepatitis E virus. After establishing the infectivity titer of the Norwalk virus inoculum, we immunized chimpanzees with recombinant viruslike particles (VLPs) consisting of the two capsid proteins of the virus. We demonstrated that such vaccinated chimpanzees were protected against challenge with live virus and that the protection was Geno group-specific. Thus, norovirus vaccines will need to be multivalent to be effective. We next plan to determine if lack of cross protection exists within Geno groups. In 2011 we collaborated with scientists in California and Japan to study the three-dimensional structure of HEV viruslike particles bound to a specific antibody. Such viruslike particles retained the antigenicity of the native HEV virion. The study revealed that the monoclonal antibodies bound to the protruding domain of the capsid proteins on the lateral side of the virion's spikes. This information will be useful for designing new hepatitis E vaccine candidates.